ABSTRACT Targeted delivery of nanocarrier contrast and drug delivery agents holds exciting potential for treating major human diseases, but new strategies are needed to maximize targeting efficiency and selectivity. A powerful attribute of nanoparticles is the ability to form multiple bonds with target cells, thereby enhancing overall adhesion strength and internalization rate. However, we currently know little about the factors that govern multivalent nanoparticle binding at the molecular level. Addressing this limitation would dramatically impact the field of targeted delivery and enable unprecedented control over multivalent nanoparticle adhesion. One of the biggest challenges is controlling targeting selectivity between normal and diseased cells that express the target molecule at different levels. Ideally the nanoparticle would display superselectivity, such that a switch-like change in binding efficiency is observed between normal and diseased cells. To date, superselectivity has only been observed in a computational model, but experimental demonstration remains a major goal in the targeting field. In previous work, we developed novel experimental methods for assessing multivalent nanoparticle adhesion dynamics and a computational simulation called Nano Adhesive Dynamics (NAD) that we used to uncover new information about bond number, dynamics, and forces. In this proposal, we will transform our experimental and simulation tools into a versatile and robust design platform that could be used to control nanoparticle binding to, and internalization within, live cells. We will use vascular inflammation, specifically the target ICAM-1, as a model system for this work due to our past experience, large inventory of affinity molecules in published literature, and connection to major diseases. Furthermore, previous work has already established the need for superselective targeting of ICAM-1. We will first add new capabilities to NAD simulations, including incorporation of the initial attachment of nanoparticles from free solution and extension of the methods to nanorods. The second phase will focus on testing molecular bond properties, with new flow chamber experiments performed using a diverse panel of anti-ICAM-1 adhesion molecules with different bond properties, as well as a class of springy peptide linkers that we hypothesize will act as molecular springs that reduce mechanical forces. The final phase of the project will be focused on adapting the work to the context of live endothelial cells, and using the NAD simulations to design and test prospective affinity molecule- nanoparticle formulations that exhibit superselective targeting behavior to normal and inflamed endothelium. The Specific Aims include: (1) advance the NAD simulation framework to model initial attachment and nanorods, (2) evaluate new molecular bond properties, (3) assess multivalent adhesion to endothelial cells, and (4) design a nanocarrier that displays superselectivity. At the conclusion of the work, we will be in ideal position to design nanocarriers that possess unique adhesive properties for targeting different diseases, with the simulation tool serving as the linchpin. This will allow us to go beyond straightforward concepts such as specificity and thermodynamics/ avidity, and instead tailor adhesion for different disease scenarios and ultimately achieve advanced behavior such as superselectivity. Obtaining this capability entirely from experiments under a guess and check format would be far too costly in terms of time, money, and effort. Furthermore, the simulation design tool will offer the versatility needed to address limitations and constrains that will be encountered under in vivo conditions. Future work will seek to validate our new targeted delivery concepts using in vivo animal models of inflammation, atherosclerosis, ischemia-reperfusion injury, and cancer.